Fig. 1: Validation of CRISPR/Cas9-mediated DAPK1 knockout in HCT116 colorectal cancer cells.

a Western Blot of DAPK1 expression status in various colorectal cancer cell lines. Representative images of two independent experiments are shown. GAPDH served as loading control. b Immunofluorescence staining of DAPK1 (green) in parental HCT116 cells and DAPK1 ko clones. Cells were counterstained with phalloidin for F-actin (red) and nuclear Hoechst (blue). Fluorescence microscopy was performed using a x 100 oil immersion objective. Representative images of two independent experiments are shown. Scale bar = 20 µm. c Protein expression of DAPK1 family members DAPK1, DAPK2, DAPK3, DRAK1, DRAK2 and DAPK1 phosphorylation target pMLC in HCT116 wildtype cells and DAPK1 ko clones were detected by Western Blotting using specific primary antibodies. Representative images of two independent experiments are shown. * images were cropped here; all samples were analyzed on the same SDS-PAGE gel; ** GAPDH blot has been used twice see Fig. 5b, proteins have been loaded on the same membrane. d Western Blot analysis of stem cell markers (CD133, CD44) and EMT markers (epithelial marker: E-cad = E-cadherin, mesenchymal marker: Vimentin). Representative images of two independent experiments are shown.*images were cropped here; all samples were analyzed on the same SDS-PAGE gel. e Representative images of endogenous phospho-ERK1/2 (red) levels of immunostained HCT116 cells and DAPK1 ko clones examined by confocal immunofluorescence microscopy (63x; enlarged: cropped and zoomed in). Cells were nuclear counterstained with Hoechst (blue). Immunofluorescence was repeated in two independent experiments and representative images are shown. White arrows: empty nucleus; dashed arrow: nuclear expression of pERK1/2. Scale bar = 50 µm. f pERK1/2 expression analyzed by Western Blot in cytoplasmic (C) and nuclear (N) protein fractions. Representative images of two independent experiments are shown. GAPDH served as loading control in total and cytoplasmatic protein fractions. Lamin A/C was used for nuclear loading control.